Dual molecules containing a peroxide derivative, synthesis and therapeutic applications thereof

ABSTRACT

The invention relates to dual molecules formed from coupling products complying with the formula  
                 
 
     wherein  
     A represents a residue of molecule with anti-malarial activity,  
     Y 1  and Y 2 , represent a linear or ramified alkylene chain at C1 to C5, with the possibility of either Y 1  or Y 2  being absent,  
     U is an amine, amide, sulphonamide, carboxyl, ether or thioether function, said function linking Y 1  and Y 2 ,  
     Z 1  and Z 2 , represent a linear arylene or alkylene, with the possibility of either Z 1  or Z 2  being absent, or Z 1 +Z 2  together represent a polycyclic structure including the junction carbons Ci and Cj,  
     R 1  and R 2 , represent a hydrogen atom or a functional group capable of increasing the hydrosolubility of the dual molecule,  
     R x  and R y  form a cyclic peroxide with 4 to 8 chain links, Cj being one of the peaks of said cyclic peroxide, or  
     R x  or R y  is a cyclic peroxide with 4 to 8 chain links, which may comprise 1 or 2 additional oxygen atoms in the cyclic structure, and one or more substituents R 3 , identical or different, at least one representing a halogen atom, an —OH group, a —CF 3  group, an aryl, an alkyl or alkoxy at C1 to C5, —NO 2 , the other substituent(s) having one of these correspondences or a hydrogen,  
     and their addition salts with pharmacological acceptable acids.  
     Application as medicinal products with anti-malarial activity.

[0001] The invention relates to dual molecules containing a peroxidederivative, showing particularly an anti-malarial activity, thesynthesis and therapeutic applications of said molecules.

[0002] Malaria is one of the primary infectious causes of mortality inthe world and affects 100 to 200 million people a year. The significantupsurge in the disease observed in recent years is due to severalfactors, including:

[0003] the carriers, i.e. Anopheles, which are becoming resistant toconventional inexpensive insecticides such as DDT (abbreviation oftrichloro-1,1,1-di(p-chloro-phenyl)-2,2 ethane);

[0004] population growth in at-risk zones and, essentially,

[0005] the resistance of numerous strains of Plasmodium falciparum, theparasite responsible for the mortal forms of the disease, to themedicinal products conventionally used, such as chloroquine andmefloquine. The discovery of artemisinine 1, 2, a powerful anti-malarialagent extracted from Artemisia annua, drew attention to moleculescomprising, like artemisinine, an endoperoxide function 3, 4.Artemisinine and some of its hemi-synthetic derivatives, such asartemether and artesunate, have proved to be very active on resistant P.falciparum strains. However, the high cost of these natural compoundsand uncertain supply represent major disadvantages. Therefore, theinterest of synthetic anti-malarial compounds, which would be accessibleat low prices, and offer an action mechanism similar to that ofartemisinine, an alkylating effect on the blood and/or parasiticproteins, will be evaluated.

[0006] Research on such compounds by the inventors led to thedevelopment of a new synthesis strategy based on the use of compoundsliable both to be accumulated effectively in the parasite and exert aneffect such as that of artemisine.

[0007] The inventors observed that forming a covalent bond between acompound with anti-malarial properties and a peroxide type derivativeoffered coupling products with, surprisingly, a synergic effect betweenthe penetration capacity and activity of the respective constituents onchloroquine-resistant strains and as a general rule a high efficacy fora wide range of parasites.

[0008] Therefore, the invention relates to dual molecules presented inthe form of coupling products, showing particularly an anti-malarialactivity, in particular on P. falciparum.

[0009] It also relates to a synthesis method for such molecules,comprising a limited number of steps, involving low-cost products, andtherefore easy to implement at an industrial scale.

[0010] The invention also relates to biological applications of saidmolecules and particularly the use of their anti-malarial properties todevelop medicinal products.

[0011] The dual molecules according to the invention are characterisedin that they consist of coupling products complying with the formula I

[0012] wherein

[0013] A represents a residue of molecule with anti-malarial activity,

[0014] Y₁ and Y₂, identical or different, represent a linear or ramifiedalkylene chain at C1 to C5, containing if applicable one or more amine,amide, sulphonamide, carboxyl, hydroxyl, ether or thioether radicals,said alkylene chain at C1 to C5 being substituted if applicable by analkyl radical at C1 to C5, with the possibility of either Y₁ or Y₂ beingabsent,

[0015] U is an amine, amide, sulphonamide, carboxyl, ether or thioetherfunction, said function linking Y₁ and Y₂,

[0016] Z₁ and Z₂, identical or different, represent a saturated orunsaturated, linear, ramified or cyclic arylene or alkylene radical,with the possibility of either Z₁ or Z₂ being absent, or Z₁+Z₂ togetherrepresent a polycyclic structure including the junction carbons Ci andCj,

[0017] R₁ and R₂, identical or different, represent a hydrogen atom or afunctional group capable of increasing the hydrosolubility of the dualmolecule, advantageously selected from —COOH, —OH, —N(R_(a), R_(b))where R_(a) and R_(b), identical or different, represent a hydrogen atomor an alkyl radical at C1 to C5,

[0018] R_(x) and R_(y) form a cyclic peroxide with 4 to 8 chain links,Cj being one of the peaks of said cyclic peroxide, or

[0019] R_(x) or R_(y) is a cyclic peroxide with 4 to 8 chain links,which may comprise 1 or 2 additional oxygen atoms in the cyclicstructure, and one or more substituents R₃, identical or different,occupying any separate positions on the cycle, at least one representinga halogen atom, an —OH group, a —CF₃ group, an aryl radical, an alkyl oralkoxy radical at C1 to C5, an —NO₂ group, the other substituent(s)having one of these correspondences or a hydrogen atom, with thepossibility of substituting the carbonated peaks of the cyclic peroxideif applicable by one or more substituents as defined for R₃, with thepossibility of two adjacent substituents forming a cyclic structure with5 to 6 chain links, saturated or unsaturated, if applicable substitutedby one or more substituents R₃ in any position, with the possibility ofthe other substituent R_(x) or R_(y) being R₃,

[0020] and their addition salts with pharmacological acceptable acids.

[0021] Advantageously, the residue A drains the coupled compound insidethe parasite, which then has an alkylating effect on the blood and/orparasitic proteins.

[0022] In a preferred family of derivatives according to the invention,A represents a nitrous heterocycle selected from an aminoquinolineaccording to formula II or a 1,5-naphtyridine according to formula IIIbelow

[0023] wherein

[0024] R₃ represents one or more identical or different substituentsoccupying separate position, at least one representing a halogen atom,an —OH group, a —CF₃ group, an aryl radical, an alkyl or alkoxy radicalat C1 to C5, an —NO₂ group, the other substituent(s) having one of thesecorrespondences or a hydrogen atom,

[0025] R₄ represents a linear, ramified or cyclic alkyl radical at C1 toC5, or a hydrogen atom.

[0026] In another preferred family according to the invention, Arepresents a radical according to formula IV

R₅—CHOH—  (IV)

[0027] wherein R₅ represents an aryl radical or a nitrous heterocyclicresidue.

[0028] Preferred correspondences for Rs consist of 9-phenanthrenyl or4-quinolinyl radicals, possibly substituted by one or more R₃ groups.

[0029] In another preferred family, A represents a phenol-2(aminomethyl)residue according to formula V

[0030] wherein R3, Ra and Rb are as defined above.

[0031] Another preferred family of dual molecules according to theinvention comprises a substituent A representing a biguanide residueselected from proguanil derivatives according to formula VI

[0032] or cycloguanil according to formula VII

[0033] wherein R₃ is as defined above.

[0034] In another preferred family, A represents a residue of pyrimidineand more particularly of pyrimethamine according to formula VIII

[0035] or formula IX

[0036] wherein R₃ is as defined above.

[0037] In another preferred family according to the invention, Arepresents an acridine residue according to formula X

[0038] wherein R₃ and R₄ are as defined above.

[0039] The invention relates to dual molecules, such as those definedabove, in particular corresponding to the preferred families mentionedabove, and wherein R_(x) and R_(y) form a cyclic peroxide together.

[0040] In more specially preferred dual molecules of this type, R_(x)and R_(y) represent a trioxane substituted by one or more substituentsR₃.

[0041] In another preferred embodiment of the invention, usedadvantageously with the previous embodiment, Z₁ and Z₂ represent acyclohexyl or bi-cyclopentyl radical.

[0042] In another preferred embodiment, used if required with at leastone of the previous embodiments, Y₁—U—Y₂ are selected so as to modulatethe hydrosolubility of the molecule to give it optimal activity.

[0043] The invention also relates to a synthesis method for themolecules defined above.

[0044] This method comprises the reaction of reactive derivatives of Aand peroxide derivatives comprising the residues R_(x) and R_(y), so asto form, between these derivatives, a link as defined in relation toformula I.

[0045] Various synthesis processes will be easy to access for thoseskilled in the art using conventional techniques. For example, forperoxide synthesis, it is possible to refer to the work by S. Pataï,“The Chemistry of peroxides”, John Wiley and Sons Ltd, 1983.

[0046] In this way, to prepare dual molecules comprising a trioxane asthe peroxide and an aminoquinoline as the derivative A, a compoundaccording to formula XI

[0047] wherein R₃ is as defined above and “hal” represents a halogenatom, is reacted with a diamine derivative according to formula XII

R₄—NH—Y₁—U₁   (XII)

[0048] where R₄ and Y₁ are as defined above and U₁ represents an —NH₂group, producing a compound according to formula XIII

[0049] wherein R₃, R₄ and Y₁ are as defined above,

[0050] b) -irradiation in the presence of molecular oxygen and aphotosensitising agent, of a derivative according to formulas XIV toXVII below

[0051] followed by the reaction with a diketone, such as1,4-cyclohexadione according to formula XVIII orcis-bicyclo(3.3.0]octane-3,7-dione according to formula XIX

[0052] producing trioxanes functionalised with a ketone, according tothe general formula XX

[0053] wherein Z₁, Z₂ and R₃ are as defined above,

[0054] c) -coupling of the derivative according to formula XIII with thetrioxane according to formula XX, by reductive amination, followed ifapplicable by a reaction with a pharmaceutically acceptable acid, toobtain the coupling product in salt form.

[0055] Step a is advantageously carried out at a temperature of 80° C.to 140° C. with stirring. The diamine derivative is preferentially usedat a rate of 5 molar equivalents. After cooling, the product obtained isrecovered by extraction, for example using an organic solvent such asdichloromethane, and then treated if required, for purificationpurposes.

[0056] To carry out step b, photosensitised oxygenation of the initialolefin is performed in the presence of molecular oxygen. Thephotosensitising agent is advantageously a conventional agent such astetra-phenylporphyrine or Bengal pink.

[0057] The peroxide obtained is then reacted with a diketone,preferentially at a rate of 4 to 10 molar equivalents. The reaction isadvantageously carried out in the presence of trimethyl silyltrifluoromethane sulphonate, at a temperature below −50° C.,particularly −70° C., for several hours. The functionalised trioxane isthen purified. Column chromatography is used for example. A similarprotocol is used for the synthesis of trioxane as the precursor oftrioxaquines according to formula XIII: 2,3-dimethylbut-2-ene isphoto-oxygenated under the above conditions and then placed in thepresence of 2 to 10 molar equivalents of an oxoaldehyde, a few drops oftrifluoroacetic acid and 2 molar equivalents of N-iodosuccinimide. Thereaction is carried out at ambient temperature and protected from lightfor several hours. The functionalised trioxane is then purified usingcolumn chromatography for example.

[0058] The coupling step c between the ketone and primary amine iscarried out in the presence of a reductive agent such as sodiumtriacetoborohydride, at ambient temperature.

[0059] These compounds are used according to a primary amine/ketonemolar ratio of approximately 1.25, the reductive agent being used at arate of 1.25 equivalents/ketone. To obtain the coupling product in saltform, the basic nitrogens undergo protonation, by adding apharmacological acceptable acid. For example, citric, tartaric, oxalicand fumaric acid may be used.

[0060] The reaction may be carried out with 2 acid equivalents. Theprotonated product is then recovered and subjected to one or morepurification steps if required.

[0061] The study of the pharmacological properties of the couplingproducts according to the invention demonstrated an anti-malarial effecton P. falciparum cultured in human red blood cells.

[0062] It is particularly important to obtain such an effect as theresistance phenomena of Plasmodium falciparum strains, the mortalspecies, are developing with respect to standard anti-malarial drugsand, in addition, vaccination protection, the subject of considerableresearch, will not be available for several years.

[0063] Therefore, the invention relates to the use of the properties ofthese coupling products, which also offer the advantage of high safety,to produce pharmaceutical formulations.

[0064] The pharmaceutical formulations according to the invention arecharacterised in that they comprise an effective quantity of at leastone coupling product as defined above, associated with apharmaceutically inert vehicle.

[0065] These formulations comprise if required active ingredients ofother medicinal products. This may involve an association with any otheranti-malarial molecule (amino-quinoline, aryl-alcohol comprising anamine function, amino derivatives of orthocresol, sulphones,sulphonamides, biguanides, amino-pyriminidines, amino-triazines, orquinazolines, and antibiotics (tetracycline, rifampicine, gramicindineD, valinomycine and quinolones in particular) and anti-fungal agentswith an anti-malarial activity).

[0066] They will also be used advantageously with compounds promotingtheir assimilation such as sugars like glucose.

[0067] The formulations according to the invention are particularlysuitable for the treatment of malaria.

[0068] Therefore, the invention also relates to the application of thecoupling products defined above to the development of medicinal productsto treat malaria.

[0069] The sales packaging materials, particularly the labelling andpackage inserts, and advantageously the packaging, are producedaccording to the planned specific therapeutic application.

[0070] The pharmaceutical formulations according to the invention may beadministered in different forms, more specifically by the oral, rectalor injectable route.

[0071] Formulations administered orally advantageously comprise 40 to300 mg of active ingredient per dosage unit, preferentially 40 to 100mg. They advantageously come in the form of tablets, pills, capsules ordrops in particular.

[0072] The injectable forms comprise 20 to 300 mg of active ingredientper dosage unit, preferentially 50 to 100 mg. They come in the form ofsolutions for injection by the intravenous, subcutaneous orintramuscular route, produced from sterile or sterilisable solutions.Suspensions or emulsions may also be used.

[0073] For rectal administration, suppositories are used.

[0074] For example, the dosage that may be used in humans corresponds tothe following doses: the patient is administered 50 to 300 mg/day forexample, in one or more doses for malaria treatment.

[0075] The invention also relates to biological reagents, wherein theactive ingredients are composed of the derivatives defined above.

[0076] These reagents may be used as references or standards in studieson potential anti-malarial activities.

[0077] The invention's other characteristics and advantages will be seenmore clearly in the following examples related to the production ofquinoline and trioxane coupling products, referred to as “trioxaquines”,and the study of their anti-parasitic activity. The formulas ofcompounds 1 to 34, the synthesis of which is described in these examplesare given at the end of the disclosure.

EXAMPLE 1

[0078] Trioxaquine 4

[0079] Synthesis of 7-chloro-4-[N-(2-aminoethyl)amino]-quinoline 1

[0080] A mixture of 4,7-dichloroquinoline (2.0 g, 10 mmol) and1,2-diaminoethane (2.7 g, 45 mmol) is heated at 85° C. for 5 hours withmagnetic stirring. After adding 1N soda (15 ml), the solid obtained isextracted with ethyl acetate (100 ml) at 50° C. The organic phase iswashed with distilled water and then using a saturated NaCl solution andthen again with distilled water and finally is dried on sodium sulphate.The solvent is evaporated and the product obtained is vacuum-dried (1.3g, 58%).

[0081] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.52 (d, ³J_(HH)=5.5 Hz, 1H,H2′), 7.94 (d, ⁴J_(HH)=2.2 Hz, 1H, H8′), 7.72 (d, ³J_(HH)=8.9 Hz, 1H,H5′), 7.35 (dd, ³J_(HH)=8.9 Hz, ⁴J_(HH)=2.2 Hz, 1H, H6′), 6.40 (d,³J_(HH)=5.5 Hz, 1H, H3′), 5.76 (s large, 1H, HN9′), 3.32 (m, 2H,H₂C10′), 3.11 (tr, 2H, H₂C11′), 1.39 (s large, H₂N12′).

[0082] MS (DCI/NH3+) m/z (%): 221 (2), 222 (MH⁺, 100), 223 (14), 224(33), 225 (4).

[0083] Synthesis of Trioxane Functionalised with a Ketone 2

[0084] A mixture of 1,4-diphenyl-1,3-cyclopentadiene (50 mg, 0.23 mmol)and tetraphenylporphyrine (5 mg) in dichloromethane (5 ml) is irradiatedin the presence of molecular oxygen (1.15 bar) for 1 hour, at 5° C.,with a white bulb (200 W). The peroxide is obtained with a quantitativeyield. The unprocessed peroxide in solution in dichloromethane is placedin a bath at −70° C.; 10 molar equivalents of 1,4-cyclohexadione (260mg, 2.3 mmol) and 0.5 equivalent of trimethylsilyl trifluoromethanesulphonate (20 μl, 0.11 mmol) are added and the reaction mixture is keptunder stirring at −70° C. for 4 hours. The reaction is stopped by addingtriethylamine (40 μl). After returning to ambient temperature, thereaction medium is washed with distilled water, dried on magnesiumsulphate and evaporated to dryness. The functionalised trioxane 2 ispurified by chromatography on silica column (hexane/ethyl acetateeluent, 80/20, v/v) (yield: 55%).

[0085] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 7.60−7.30 (m, 10H, H-phenyl),6.35 (d, J_(HH)=1.6 and 4.0 Hz, 1H, H6), 5.26 (s large, 1H, H5), 3.31and 3.05 (2×d, ²J_(HH)=17.0 Hz, 2×1H, H₂C6), 2.56−2.43 (m, 5H), 2.26 (m,1H), 2.05 (m, 2H).

[0086] MS (DCI/NH3+) m/z (6): 363 (MH⁺, 24), 364 (7), 380 (MNH₄ ⁺, 100),381 (27), 382 (7).

[0087] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquine 3

[0088] The ketone 2 (99 mg, 0.27 mmol) and the primary amine 1 (76 mg,0.34 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (72 mg, 0.34 mmol) is added. The mixture is keptunder stirring at ambient temperature for 18 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 87%).

[0089] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (2×d, 1H, H2′), 7.95 (2×d,1H, H8′), 7.70 (2×d, 1H, H5′), 7.63−7.25 (m, 11H, H6′ and 10H phenyl),6.35 (m, 2H, H3′ and H6), 5.99 (s large, 1H, HN9′), 5.17 (2×s large, 1H,H5), 3.31 (m, 3H, H₂C10′ and HC8), 3.05 (m, 3H, H₂C11′ and HC8), 2.61(m, 2H, cyclohexyl), 2.42 (m, 1H, HC12), 2.10−1.25 (m, 7H, 6H,cyclohexyl and HN12′).

[0090] MS (DCI/NH3+) m/z (%): 566 (11), 568 (MH⁺, 100), 569 (38), 570(41), 571 (12).

[0091] Production of Trioxaquine Dicitrate 4

[0092] The trioxaquine 3 (25 mg, 0.04 mmol) is placed in solution inacetone (0.5 ml). Citric acid (17 mg, 2.0 equiv.) in solution in acetone(0.5 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0093] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.59 (2×d, 1H, H2′), 8.30 (2×d,1H, H5′), 7.95 (2×d, 1H, H8′), 7.70 (m, 5H, H6′ and 4H phenyl), 7.50 (m,6H, phenyl), 6.73 (2×d, 1H, H3′), 6.60 (2×q, 1H, H6), 5.42 (2×s large,1H, H5), 3.71 (m, 2H, H₂C10′), 3.55−3.25 (m, 4H, H₂C11′, HC8 and HC12),3.12 (d, 1H, HC8), 2.76 (d, 4H, citrate), 2.65 (d, 4H, citrate),2.10−1.50 (m, 8H, cyclohexyl).

[0094] MS (ES) m/z (%):

[0095] in positive mode 568.2 (M⁺)

[0096] in negative mode 190.9 (citrate)

[0097] Elementary microanalysis: for C₄₆H₅₀O₁₇N₃Cl Theor. %: C 58.01 H5.29 N 4.41 Exper. %: C 57.65 H 5.09 N 4.49

EXAMPLE 2

[0098] Trioxaquine 7

[0099] Synthesis of 7-chloro-4-[N-(3-aminopropyl)amino]-quinoline 5

[0100] A mixture of 4,7-dichloroquinoline (5 g, 25 mmol) and1,3-diaminopropane (9.3 g, 126 mmol) is diamine reflux-heated (118° C.)for 5 hours with magnetic stirring. After cooling, the solid obtained isreflux-extracted with dichloromethane (3×100 ml). The organic phase iswashed with distilled water and then dried on sodium sulphate. Theconcentration of the dichloromethane phase followed by the addition ofhexane precipitate the product in the form of a light yellow solid whichis filtered, washed with hexane and vacuum-dried (4.5 g, yield=76%).

[0101] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.48 (d, ³J_(HH)=5.5 Hz, 1H,H2′), 7.90 (d, ⁴J_(HH)=2.2 Hz, 1H, H8′), 7.70 (d, ³J_(HH)=8.9 Hz, 1H,H5′), 7.50 (s large, 1H, HN9′), 7.29 (dd, ³J_(HH)=8.9 Hz, ⁴J_(HH)=2.2Hz, 1H, H6′), 6.30 (d, ³J_(HH)=5.5 Hz, 1H, H3′), 3.39 (m, 2H, H₂C10′),3.03 (tr, 2H, H₂C11′), 1.87 (m, 2H, H₂C11′), 1.58 (s large, H₂N13′).

[0102] MS (DCI/NH3+) m/z (%): 235 (2), 236 (MH⁺, 100), 237 (14), 238(34), 239 (5).

[0103] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquine 6

[0104] The ketone 2 (199 mg, 0.55 mmol) and the primary amine 5 (165 mg,0.70 mmol) are placed in solution in CH₂Cl₂ (10 ml). Sodiumtriacetoxyborohydride (146 mg, 0.69 mmol) is added. The mixture is keptunder stirring at ambient temperature for 15 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 96%).

[0105] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.51 (2×d, 1H, H2′), 8.04 (slarge, 1H, HN9′), 7.90 (2×d, 1H, H8′), 7.80 (2×d, 1H, H5′), 7.65−7.25(m, 11H, H6′ and 10H phenyl), 6.30 (m, 2H, H3′ and H6), 5.14 (2×s large,1H, H5), 3.39 (q, 2H, H₂C10′), 3.29 (d, 1H, HC8), 2.95 (m, 3H, H₂C12′and HC8), 2.60 (m, 2H, cyclohexyl), 2.42 (m, 2H, HC12 and HN13′),2.10−1.25 (m, 10H, 8H, cyclohexyl and H₂C11′).

[0106] MS (DCI/NH3+) m/z (%): 580 (5), 582 (MH⁺, 100) 583 (39), 584(39), 585 (15).

[0107] Production of Trioxaquine Dicitrate 7

[0108] The trioxaquine 4 (81 mg, 0.14 mmol) is placed in solution inacetone (4 ml). Citric acid (80 mg, 3.0 equiv.) in solution in acetone(5 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0109] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.60 (2×d, 1H, H2′), 8.42 (2×d,1H, H5′), 7.93 (2×d, 1H, H8′), 7.65 (m, 5H, H6′ and 4H phenyl), 7.45 (m,6H, phenyl), 6.72 (2×d, 1H, H3′), 6.61 (2×q, 1H, H6), 5.43 (2×s large,1H, H5), 3.8−3.0 (m, 7H, H₂C10′, H₂C12′, HC8 and HC12), 2.76 (d, 4H,citrate), 2.65 (d, 4H, citrate), 2.20−1.40 (m, 10H, 8H cyclohexyl andH₂C11′).

[0110] MS (ES) m/z (%):

[0111] in positive mode 582.3 (M⁺)

[0112] in negative mode 190.8 (citrate)

[0113] Elementary microanalysis: for C₄₇H₅₂O₁₇N₃Cl, 1 H₂O Theor. %: C57.35 H 5.53 N 4.27 Exper. %: C 57.09 H 5.20 N 4.24

EXAMPLE 3

[0114] Trioxaquine 10

[0115] Synthesis of 7-chloro-4-[N-(4-aminobutyl)amino]-quinoline 8

[0116] A mixture of 4,7-dichloroquinoline (5 g, 25 mmol) and1,4-diaminobutane (13 ml, 129 mmol) is diamine reflux-heated for 5 hourswith magnetic stirring. After cooling, the solid obtained isreflux-extracted with dichloromethane (3×100 ml). The organic phase iswashed with distilled water and then dried on sodium sulphate. Theconcentration of the dichloromethane phase followed by the addition ofhexane precipitate the product in the form of a light yellow solid whichis filtered, washed with hexane and vacuum-dried (3.4 g, yield=54%).

[0117] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (d, ³J_(HH)=5.5 Hz, 1H,H2′), 7.92 (d, ⁴J_(HH)=2.2 Hz, 1H, H8′), 7.72 (d, ³J_(HH)=8.9 Hz, 1H,H5′), 7.31 (dd, ³J_(HH)=8.9 Hz, ⁴J_(HH)=2.2 Hz, 1H, H6′), 6.36 (d,³J_(HH)=5.5 Hz, 1H, H3′), 6.04 (s large, 1H, HN9′), 3.29 (m, 2H,H₂C10′), 2.81 (tr, 2H, H₂C13′), 1.85 (m, 2H, H₂C11′), 1.64 (m, 2H,H₂C12′), 1.45 (s large, H₂N14′).

[0118] MS (DCI/NH3+) m/z (%): 249 (2), 250 (MH⁺, 100), 251 (18), 252(36), 253 (5).

[0119] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquine 9

[0120] The ketone 2 (170 mg, 0.47 mmol) and the primary amine 8 (150 mg,0.60 mmol) are placed in solution in CH₂Cl₂ (10 ml). Sodiumtriacetoxyborohydride (125 mg, 0.59 mmol) is added. The mixture is keptunder stirring at ambient temperature for 15 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 69%).

[0121] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (2×d, 1H, H2′), 7.89 (2×d,1H, H8′), 7.78 (2×d, 1H, H5′), 7.65−7.25 (m, 11H, H6′ and 10H phenyl),6.35 (m, 2H, H3′ and H6), 5.96 (s large, 1H, HN9′), 5.18 (2×s large, 1H,H5), 3.30 (m, 3H, H₂C10′ and HC8), 3.00 (2×d, 1H, HC8), 2.74 (q, 2H,H₂C13′), 2.61 (m, 2H, cyclohexyl), 2.46 (m, 1H, HC12), 2.10−1.25 (m,11H, 6H, cyclohexyl HN14′, H₂C11′ and H₂C12′).

[0122] MS (DCI/NH3+) m/z (%): 596 (MH⁺).

[0123] Production of Trioxaquine Dicitrate 10

[0124] The trioxaquine 9 (51 mg, 0.09 mmol) is placed in solution inacetone (1 ml). Citric acid (33 mg, 2.0 equiv.) in solution in acetone(1 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0125] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.56 (2×d, 1H, H2′), 8.45 (2×d,1H, H5′), 7.95 (m+2×d, 2H, HN9′ and H8′), 7.68 (m, 5H, H6′ and 4Hphenyl), 7.48 (m, 6H, phenyl), 6.72 (2×d, 1H, H3′), 6.60 (2×q, 1H, H6),5.41 (2×s large, 1H, H5), 3.50 (m, 2H, H₂C10′), 3.55−3.10 (m, 5H,H₂C13′, H₂C8′ and HC12), 2.75 (d, 4H, citrate), 2.65 (d, 4H, citrate),2.10−1.50 (12H, 8H cyclohexyl, H₂C11′ and H₂C12′).

[0126] MS (ES) m/z (%):

[0127] in positive mode 596.2 (M⁺)

[0128] in negative mode 190.8 (citrate)

[0129] Elementary microanalysis: for C₄₈H₅₄O₁₇N₃Cl, 4 H₂O Theor. %: C54.78 H 5.94 N 3.99 Exper. %: C 54.88 H 5.08 N 4.06

EXAMPLE 4

[0130] Trioxaquines 13a and 13b

[0131] Synthesis of Trioxane Functionalised with a Ketone 11

[0132] A mixture of 1,4-diphenyl-1,3-cyclopentadiene (153 mg, 0.7 mmol)and tetraphenylporphyrine (5 mg) in dichloromethane (5 ml) is irradiatedin the presence of molecular oxygen (1.15 bar) for 1 hour, at 5° C.,with a white bulb (200 W). The peroxide is obtained with a quantitativeyield. The unprocessed peroxide in solution in dichloromethane is placedin a bath at −70° C.; 4 molar equivalents ofcisbicyclo(3.3.0)octane-3,7-dione (410 mg, 3.0 mmol) and 0.4 equivalentof trimethylsilyl trifluoromethane sulphonate (50 μl, 0.3 mmol) areadded and the reaction mixture is kept under stirring at −70° C. for 2hours. The reaction is stopped by adding triethylamine (100 μl). Afterreturning to ambient temperature, the reaction medium is washed withdistilled water, dried on magnesium sulphate and evaporated to dryness.Chromatography on silica column (hexane/ethyl acetate eluent, 70/30,v/v) is used to separate the two isomer trioxanes 11a and 11b (overallyield: 42%).

[0133] Isomer 11a:

[0134] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 7.60−7.30 (m, 10H, phenyl), 6.29(dd, 1H, H6), 5.27 (s large, 1H, H5), 3.21 and 3.02 (2×d, ²J_(HH)=17.0Hz, 2×1H, H₂C8), 2.83 (m, 2H), 2.20 (m, 3H), 1.77 (m, 2H).

[0135] MS (DCI/NH3+) m/z (%): 406 (MNH₄ ⁺, 100), 407 (30), 408 (8).

[0136] Isomer 11b:

[0137] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 7.60−7.30 (m, 10H, phenyl), 6.32(dd, 1H, H6), 5.25 (s large, 1H, H5), 3.22 and 3.02 (2×d, ²J_(HH)=17.0Hz, 2×1H, H₂C8), 2.90 (m, 2H), 2.50−2.15 (m, 7H), 1.79 (m, 1H).

[0138] MS (DCI/NH3+) m/z (%): 404 (3), 405 (3), 406 (MNH₄ ⁺, 100), 407(31), 408 (6), 409 (1).

[0139] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquines 12a and 12b

[0140] The ketone 11a (163 mg, 0.42 mmol) and the primary amine 1 (120mg, 0.54 mmol) are placed in solution in CH₂Cl₂ (15 ml). Sodiumtriacetoxyborohydride (114 mg, 0.54 mmol) is added. The mixture is keptunder stirring at ambient temperature for several weeks. The reactionmedium is then washed with distilled water; the organic phase is driedand the solvent is evaporated to dryness (yield: 66%).

[0141] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.42 (d, 1H, H2′), 7.86 (d, 1H,H8′), 7.75 (d, 1H, H5′), 7.65−7.25 (m, 11H, H6′ and 10H phenyl), 6.30(m, 1H, H3′), 6.23 (m, 1H, H6), 6.18 (s large, 1H, HN9′), 5.25 (s large,1H, H5), 3.57 (s large, 1H, HN12′), 3.37 (m, 3H, H₂C10′, HC8), 3.20 (m,2H, HC8 and 1H bicyclopentyl), 3.00 (m, 3H, H₂C11′ and HC8), 2.75 (m,1H, bicyclopentyl), 2.45 (m, 2H, HC12 and 1H bicyclopentyl), 2.20 (m,2H, bicyclopentyl), 1.74−1.10 (m, 5H, bicyclopentyl).

[0142] MS (DCI/NH3+) m/z (%): 594 (MH⁺).

[0143] The ketone 11b (148 mg, 0.38 mmol) and the primary amine 1 (110mg, 0.50 mmol) are placed in solution in CH₂Cl₂ (15 ml). Sodiumtriacetoxyborohydride (154 mg, 0.73 mmol) is added. The mixture is keptunder stirring at ambient temperature for one week. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 68%).

[0144] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.48 (d, 1H, H2′), 7.88 (d, 1H,H8′), 7.73 (d, 1H, H5′), 7.65−7.25 (m, 11H, H6′ and 10H phenyl), 6.27(m, 1H, H3′ and H6), 6.06 (s large, 1H, HN9′), 5.25 (s large, 1H, H5),3.25 (m, 2H, H₂C10′), 3.21 (d, 1H, HC8), 3.01 (d, 1H, HC8), 2.94 (m, 3H,H₂C11′ and 3H bicyclopentyl), 2.54 (m, 3H, HC12 and 2H bicyclopentyl),2.10 (m, 4H, HN12′ and 3H bicyclopentyl), 1.77 (m, 1H, bicyclopentyl),1.25 (m, 3H, bicyclopentyl).

[0145] MS (DCI/NH3+) m/z (%): 594 (MH⁺, 100), 595 (44), 596 (44).

[0146] Production of Trioxaquine Dicitrate 13a and 13b

[0147] The trioxaquine 12a (166 mg, 0.28 mmol) is placed in solution inacetone (5 ml). Citric acid (160 mg, 3.0 equiv.) in solution in acetone(5 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0148] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.63 (d, 1H, H2′), 8.40 (d, 1H,H5′), 8.00 (d, 1H, H8′), 7.70 (m, 5H, H6′ S and 4H phenyl), 7.50 (m, 6H,phenyl), 6.79 (d, 1H, H3′), 6.60 (q, 1H, H6), 5.53 (s large, 1H, H5),3.75 (m, 3H, H₂C10′ and HC8), 3.30 (m, 2H, HC8 and HC12), 3.12 (m, 2H,H₂C11′), 2.68 (d, 4H, citrate), 2.39 (m, 4H, citrate), 2.10−1.50 (m, 6H,bicyclopentyl).

[0149] MS (ES) m/z (%):

[0150] in positive mode 594.3 (M⁺)

[0151] Elementary microanalysis: for C₄₈H₅₂O₁₇N₃Cl, 1 H₂O Theor. %: C57.86 H 5.46 N 4.22 Exper. %: C 58.11 H 5.02 N 4.66

[0152] The trioxaquine 12b (153 mg, 0.26 mmol) is placed in solution inacetone (5 ml). Citric acid (160 mg, 3.0 equiv.) in solution in acetone(5 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0153] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.60 (d, 1H, H2′), 8.34 (d, 1H,H5′), 7.95 (d, 1H, H8′), 7.73 (m, 5H, H6′ and 4H phenyl), 7.51 (m, 6H,phenyl), 6.73 (d, 1H, H3′), 6.60 (q, 1H, H6), 5.55 (s large, 1H, H5),3.69 (m, 3H, H₂C10′ and HC8), 3.44 (m, 1H, HC12), 3.25 (m, 1H, HC8),3.16 (m, 2H, H₂C11′), 2.77 (d, 4H, citrate), 2.65 (m, 4H, citrate),2.55−2.05 (m, 6H, bicyclopentyl), 1.80−1.40 (m, 4H, bicyclopentyl).

[0154] MS (ES) m/z (%):

[0155] in positive mode 594.3 (M⁺)

[0156] in negative mode 190.9 (citrate)

[0157] Elementary microanalysis: for C₄₈H₅₂O₁₇N₃Cl, 1 H₂O Theor. %: C57.86 H 5.46 N 4.22 Exper. %: C 58.19 H 5.05 N 4.17

EXAMPLE 5

[0158] Trioxaquines 15a and 15b

[0159] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquines 14a and 14b

[0160] The ketone 11a (29 mg, 0.075 mmol) and the primary amine 8 (25mg, 0.10 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (21 mg, 0.10 mmol) is added. The mixture is keptunder stirring at ambient temperature for 48 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 64%).

[0161] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.45 (d, 1H, H2′), 7.92 (d, 1H,H8′), 7.75 (d, 1H, H5′), 7.65−7.25 (m, 11H, H6′ and 10H phenyl), 6.33(m, 2H, H3′ and HN9′), 6.27 (m, 1H, H6), 5.25 (s large, 1H, H5), 3.25(m, 2H, H₂C10′), 3.19 (d, 1H, HC8), 3.00 (m, 2H, HC8 and 1Hbicyclopentyl), 2.8−2.0 (m, 8H, 4H bicyclopentyl, H₂C13′, HC12 andHN14′), 1.75−1.10 (m, 5H bicyclopentyl, H₂C11′ and H₂C12′).

[0162] MS (DCI/NH3+) m/z (%): 622 (MH⁺).

[0163] The ketone 11b (26 mg, 0.070 mmol) and the primary amine 8 (21mg, 0.084 mmol) are placed in solution in CH₂Cl₂ (15 ml). Sodiumtriacetoxyborohydride (18 mg, 0.085 mmol) is added. The mixture is keptunder stirring at ambient temperature for 48 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness. The mixture obtained contains 70%trioxaquine 12b and is used as is in the next step.

[0164] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.44 (d, 1H, H2′), 7.90 (d, 1H,H8′), 7.75 (d, 1H, H5′), 7.65−7.25 (m, 11H, H6′and 10H phenyl), 6.30 (m,3H, H3′, H6 and HN9′), 5.24 (s large, 1H, H5), 3.25 (m, 2H, H₂C10′),3.20 (d, 1H, HC8), 3.00 (m, 1H, HC8), 2.85 (m, 1H, bicyclopentyl),2.65−2.0 (m, 9H, 5H bicyclopentyl, H₂C13′, HC12 and HN14′), 1.8−1.6 (m,5H bicyclopentyl, 1H bicyclopentyl, H₂C11′ and H₂C12′), 1.24 (m, 3H,bicyclopentyl).

[0165] Production of Trioxaquine Dicitrates 15a and 15b

[0166] The trioxaquine 14a (30 mg, 0.05 mmol) is placed in solution inacetone (0.4 ml). Citric acid (22 mg, 2.4 equiv.) in solution in acetone(0.4 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0167] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.59 (d, 1H, H2′), 8.48 (d, 1H,H5′), 8.17 (s large, 1H, HN9′), 7.97 (d, 1H, H8′), 7.70 (m, 5H, H6′ and4H phenyl), 7.50 (m, 6H, phenyl), 6.77 (d, 1H, H3′), 6.57 (q, 1H, H6),5.50 (s large, 1H, H5), 3.51 (m, 2H, H₂C10′ and HC8), 3.10 (m, 4H,H₂C13′, HC8 and HC12), 2.78 (d, 4H, citrate), 2.67 (m, 4H, citrate),2.37 (m, 4H, bicyclopentyl), 2.10−1.50 (m, 10H, 6H bicyclopentyl, H₂C11′and H₂C12′).

[0168] MS (ES) m/z (%):

[0169] in positive mode 622.3 (M⁺)

[0170] in negative mode 191.2 (citrate)

[0171] The trioxaquine 14b (28 mg, unprocessed mixture) is placed insolution in acetone (1 ml). Citric acid (30 mg, 4.0 equiv.) in solutionin acetone (2 ml) is added. The trioxaquine dicitrate precipitates; itis centrifuged, washed twice in diethyl ether and vacuum-dried.

[0172] NMR ¹H (250 MHz, DMSO-d6) δ, ppm: 8.57 (d, 1H, H2′), 8.45 (d, 1H,H5 ′), 7.93 (d, 1H, H8′), 7.70 (m, 5H, H6′ and 4H phenyl), 7.51 (m, 6H,phenyl), 6.70 (d, 1H, H3′), 6.61 (q, 1H, H6), 5.52 (s large, 1H, H5),4.0−3.0 (m, 7H, H₂C11′, H₂C13′, H₂C8 and HC12), 2.75 (d, 4H, citrate),2.65 (m, 4H, citrate), 2.55−2.05 (m, 6H, bicyclopentyl), 1.90−1.30 (m,8H, 4H bicyclopentyl, H₂C11′ and H₂C12′).

[0173] MS (ES) m/z (%):

[0174] in positive mode 622.4 (M⁺)

[0175] in negative mode 191.2 (citrate)

EXAMPLE 6

[0176] Trioxaquine 18

[0177] Synthesis of Trioxane Functionalised with a Ketone 16

[0178] A mixture of α-terpinene (420 mg, 3.0 mmol) andtetraphenylporphyrine (5 mg) in dichloromethane (5 ml) is irradiated inthe presence of molecular oxygen (1.15 bar) for 7 hours, at 5° C., witha white bulb (200 W). The peroxide is obtained with a quantitativeyield. The unprocessed peroxide in solution in dichloromethane is placedin a bath at −70° C.; 6 molar equivalents of 1,4-cyclohexadione (2.05 g,18.3 mmol) and 0.4 equivalent of trimethylsilyl trifluoromethanesulphonate (200 μl, 1.1 mmol) are added and the reaction mixture is keptunder stirring at −70° C. for 2 hours. The reaction is stopped by addingtriethylamine (400 μl). After returning to ambient temperature, thereaction medium is washed with distilled water, dried on magnesiumsulphate and evaporated to dryness. The functionalised trioxane 16 ispurified by chromatography on silica column (hexane/ethyl acetateeluent, 85/15, v/v) (yield: 38%).

[0179] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 5.40 (m, 1H, H6), 4.00 (m, 1H,H5), 2.67 (m, 1H), 2.41 (m, 4H), 2.22 (m, 4H), 2.00 (m, 3H), 1.50 (m,1H), 0.99 (m, 9H).

[0180] MS (DCI/NH3+) m/z (%): 297 (26), 298 (MNH₄ ⁺, 100), 299 (48), 300(8), 301 (1).

[0181] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquine 17

[0182] The ketone 16 (113 mg, 0.40 mmol) and the primary amine 1 (115mg, 0.52 mmol) are placed in solution in CH₂Cl₂ (10 ml). Sodiumtriacetoxyborohydride (109 mg, 0.51 mmol) is added. The mixture is keptunder stirring at ambient temperature for 20 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 82%).

[0183] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (2×d, 1H, H2′), 7.92 (2×d,1H, H8′), 7.69 (2×d, 1H, H5′), 7.35 (2×dd, 1H, H6′), 6.36 (2×d, 1H,H3′), 5.95 (s large, 1H, HN9′), 5.41 (m, 1H, H6), 4.00 (m, 1H, H5), 3.29(m, 2H, H₂C10′), 3.02 (m, 2H, H₂C11′), 2.63 (m, 2H), 2.43 (m, 1H),2.20−1.90 (m, 5H), 1.85 (m, 5H), 1.50 (m, 4H), 1.02 (m, 9H).

[0184] MS (DCI/NH3+) m/z (%): 484 (2), 485 (6), 486 (MH⁺, 100), 487(36), 488 (42), 489 (12), 490 (2).

[0185] Production of Trioxaquine Dicitrate 18

[0186] The trioxaquine 17 (45 mg, 0.09 mmol) is placed in solution inacetone (1 ml). Citric acid (53 mg, 3.0 equiv.) in solution in acetone(1 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0187] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.62 (2×d, 1H, H2′), 8.35 (2×d,1H, H5′), 7.97 (2×d, 1H, H8′), 7.69 (2×dd, 1H, H6′), 6.75 (2×d, 1H,H3′), 5.45 (m, 1H, H6), 4.17 (m, 1H, H5), 3.75 (m, 2H, H₂C10′), 3.35 (m,2H, H₂C11′), 3.05 (m, 1H), 2.76 (d, 4H, citrate), 2.65 (d, 4H, citrate),2.29 (m, 3H), 2.07 (m, 4H), 1.72 (m, 3H), 1.57 (m, 3H), 1.10 (m, 9H).

[0188] MS (ES) m/z (%):

[0189] in positive mode 486.2 (M⁺) Theor. %: C 53.84 H 6.03 N 4.83Exper. %: C 54.25 H 5.43 N 5.04

EXAMPLE 7

[0190] Trioxaquine 20

[0191] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquine 19

[0192] The ketone 16 (100 mg, 0.36 mmol) and the primary amine 8 (115mg, 0.46 mmol) are placed in solution in CH₂Cl₂ (10 ml). Sodiumtriacetoxyborohydride (101 mg, 0.48 mmol) is added. The mixture is keptunder stirring at ambient temperature for 15 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 62%).

[0193] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.47 (2×d, 1H, H2′), 7.89 (2×d,1H, H8′), 7.74 (2×d, 1H, H5′), 7.35 (2×dd, 1H, H6′), 6.34 (2×d, 1H,H3′), 5.9−5.7 (m, 1H, HN9′), 5.39 (m, 1H, H6), 4.00 (m, 1H, H5), 3.27(m, 2 H, H₂C10′), 2.70 (m, 2 H, H₂C13′), 2.60−2.35 (m, 3H), 2.30−1.95(m, 5H), 1.83 (m, 3H), 1.67 (m, 4H, H₂C11′ and H₂C12′), 1.50 (m, 4H),1.00 (m, 9H).

[0194] MS (DCI/NH3+) m/z (%): 514 (3), 515 (MH⁺, 100), 516 (28), 517(36), 518 (11).

[0195] Production of Trioxaquine Dicitrate 20

[0196] The trioxaquine 19 (46 mg, 0.09 mmol) is placed in solution inacetone (1 ml). Citric acid (44 mg, 2.6 equiv.) in solution in acetone(1 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0197] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.56 (2×d, 1H, H2′), 8.45 (2×d,1H, H5′), 7.95 (2×d, 1H, H8′), 7.67 (2×dd, 1H, H6′), 6.72 (2×d, 1H,H3′), 5.45 (m, 1H, H6), 4.15 (m, 1H, H5), 3.50 (m, 2H, H₂C10′), 3.20 (m,1H), 3.05 (m, 2H, H₂C13′), 2.76 (d, 4H, citrate), 2.65 (d, 4H, citrate),2.29 (m, 3H), 2.07 (m, 4H), 1.80 (m, 7H), 1.55 (m, 3H), 1.10 (m, 9H).

[0198] MS (ES) m/z (%):

[0199] in positive mode 514.4 (M⁺)

[0200] Elementary microanalysis: for C₄₁H₅₆O₁₇N₃Cl Theor. %: C 54.83 H6.29 N 4.68 Exper. %: C 54.32 H 5.80 N 4.52

EXAMPLE 8

[0201] Trioxaquines 23a and 23b

[0202] Synthesis of Trioxanes Functionalised with a Ketone 21a, 21b and21c

[0203] A mixture of α-terpinene (320 mg, 2.76 mmol) andtetraphenylporphyrine (5 mg) in dichloromethane (10 ml) is irradiated inthe presence of molecular oxygen (1.15 bar) for 7 hours, at 5° C., witha white bulb (200 W). The peroxide is obtained with a quantitativeyield. The unprocessed peroxide in solution in dichloromethane is placedin a bath at −70° C.; 4 molar equivalents ofcisbicyclo(3.3.0)octane-3,7-dione (1.62 g, 11.7 mmol) and 0.5 equivalentof trimethylsilyl trifluoromethane sulphonate (250 μl, 1.38 mmol) areadded and the reaction mixture is kept under stirring at −70° C. for 4hours. The reaction is stopped by adding triethylamine (400 μl). Afterreturning to ambient temperature, the reaction medium is washed withdistilled water, dried on magnesium sulphate and evaporated to dryness.Chromatography on silica column (hexane/ethyl acetate eluent, 70/30,v/v) is used to separate three isomer trioxanes 21a, 21b and 21c, inorder of elution (overall yield: 35%).

[0204] Isomer 21a:

[0205] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 5.37 (d large, 1H, H6), 3.85 (dlarge, 1H, H5), 3.10−2.60 (m, 3H), 2.48 (m, 2H), 2.16 (m, 6H), 1.90−1.40(m, 4H), 0.99 (m, 9H).

[0206] MS (DCI/NH3+) m/z (): 323 (4), 324 (MNH₄ ⁺, 100), 325 (21), 326(5).

[0207] Isomer 21b:

[0208] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 5.38 (d large, 1H, H6), 3.88 (dlarge, 1H, H5), 2.82 (m, 2H), 2.62 (m, 1H), 2.5−2.0 (m, 10H), 1.72 (m,1H), 1.49 (m, 1H), 0.99 (m, 9H).

[0209] MS (DCI/NH3+) m/z (%): 323 (14), 324 (MNH₄ ⁺, 100), 325 (23), 326(5), 327 (1).

[0210] Isomer 21c:

[0211] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 5.23 (m, 1H, H6), 4.40 (m, 1H,H5), 3.10−2.6 (m, 3H), 2.49 (m, 2H), 2.17 (m, 6H), 1.9−1.5 (m, 4H), 1.37(s, 3H), 1.00 (m, 6H).

[0212] MS (DCI/NH3+) m/z (%): 323 (26), 324 (MNH₄ ⁺, 100) 325 (19), 326(5), 327 (1).

[0213] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquines 22a and 22b

[0214] The ketone 21a (70 mg, 0.23 mmol) and the primary amine 1 (66 mg,0.30 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (61 mg, 0.29 mmol) is added. The mixture is keptunder stirring at ambient temperature, for one week. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 70%).

[0215] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.45 (d, 1H, H2′), 7.85 (d, 1H,H8′), 7.70 (d, 1H, H5′), 7.29 (dd, 1H, H6′), 6.31 (d, 1H, H3′), 6.09 (slarge, 1H, HN9′), 5.36 (d large, 1H, H6), 3.87 (d large, 1H, H5), 3.31(m, 2H, H₂C10′), 3.11 (m, 2H), 2.99 (m, 2H, H₂C11′), 2.75−2.35 (m, 3H),2.20 (m, 6H), 1.90−1.40 (m, 4H), 1.21 (m, 2H), 0.98 (m, 9H).

[0216] MS (DCI/NH3+) m/z (%) 512 (MH⁺, 100), 513 (32), 514 (46), 515(15), 516 (7).

[0217] The ketone 21b (35 mg, 0.11 mmol) and the primary amine 1 (32 mg,0.14 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (30 mg, 0.14 mmol) is added. The mixture is keptunder stirring at ambient temperature for several weeks. The reactionmedium is then washed with distilled water; the organic phase is driedand the solvent is evaporated to dryness (yield: 75%).

[0218] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.46 (d, 1H, H2′), 7.90 (d, 1H,H8′), 7.76 (d, 1H, H5′), 7.35 (dd, 1H, H6′), 6.34 (d, 1H, H3′), 6.03 (slarge, 1H, HN9′), 5.42 (d large, 1H, H6), 3.89 (d large, 1H, H5), 3.26(m, 2H, H₂C10′), 2.97 (m, 3H, H₂C11′ and 1H), 2.7−1.9 (m, 11H), 1.70 (m,2H), 1.50 (m, 2H), 1.24 (m, 2H), 0.98 (m, 9H).

[0219] MS (DCI/NH3+) m/z (%): 510 (10), 512 (MH⁺, 100), 513 (35), 514(51), 515 (16), 516 (7).

[0220] Production of Trioxaquine Dicitrates 23a and 23b

[0221] The trioxaquine 22a (82 mg, 0.16 mmol) is placed in solution inacetone (5 ml). Citric acid (90 mg, 2.9 equiv.) in solution in acetone(5 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0222] NMR ¹H (250 MHz, DMSO-d6) δ, ppm: 8.62 (d, 1H, H2′), 8.40 (d, 1H,H5′), 7.97 (d, 1H, H8′), 7.69 (dd, 1H, H6′), 6.78 (d, 1H, H3′), 5.49 (dlarge, 1H, H6), 4.02 (d large, 1H, H5), 3.78 (m, 2H, H₂C10′), 3.65 (m,1H), 3.32 (m, 2H, H2C11′), 2.79 (d, 4H, citrate), 2.68 (d, 4H, citrate),2.60−1.90 (m, 9H), 1.80 (m, 4H), 1.10 (m, 9H).

[0223] MS (ES) m/z (%):

[0224] in positive mode 512.3 (M⁺)

[0225] in negative mode 190.8 (citrate)

[0226] Elementary microanalysis: for C₄₁H₅₄O₁₇N₃Cl Theor. %: C 54.95 H6.08 N 4.69 Exper. %: C 55.07 H 6.15 N 4.52

[0227] The trioxaquine 22b (44 mg, 0.09 mmol) is placed in solution inacetone (4 ml). Citric acid (50 mg, 3.0 equiv.) in solution in acetone(4 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0228] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.62 (d, 1H, H2′), 8.36 (d, 1H,H5′), 7.96 (d, 1H, H8′), 7.70 (dd, 1H, H6′), 6.75 (d, 1H, H3′), 5.48 (dlarge, 1H, H6), 4.05 (d large, 1H, H5), 3.73 (m, 2H, H₂C10′), 3.45 (m,1H), 3.29 (m, 2H, H₂C11′), 2.80 (d, 4H, citrate), 2.68 (d, 4H, citrate),2.31 (m, 6H), 2.06 (m, 4H), 1.8−1.4 (m, 5H), 1.10 (m, 9H).

[0229] MS (ES) m/z (%):

[0230] in positive mode 512.5 (M⁺)

[0231] in negative mode 191.2 (citrate)

[0232] Elementary microanalysis: for C₄₁H₅₄O₁₇N₃Cl, 2 H₂O Theor. %: C52.81 H 6.27 N 4.51 Exper. %: C 52.93 H 5.49 N 5.18

EXAMPLE 9

[0233] Trioxaquines 25a and 25b

[0234] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquines 24a and 24b

[0235] The ketone 21a (70 mg, 0.23 mmol) and the primary amine 8 (71 mg,0.28 mmol) are placed in solution in H₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (61 mg, 0.29 mmol) is added. The mixture is keptunder stirring at ambient temperature for one week. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 51%).

[0236] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.49 (d, 1H, H2′), 7.92 (d, 1H,H8′), 7.74 (d, 1H, H5′), 7.32 (m, 1H, H6′), 6.35 (m, 1H, H3′), 6.0−5.8(m, 1H, HN9′), 5.39 (d large, 1H, H6), 3.88 (d large, 1H, H5), 3.26 (m,2H, H₂C10′), 2.91 (m, 2H), 2.68 (m, 2H, H₂C11′), 2.48 (m, 3H), 2.20 (m,6H), 1.90−1.40 (m, 8H), 1.22 (m, 2H), 0.97 (m, 9H).

[0237] MS (DCI/NH3+) m/z (%): 538 (7), 539 (5), 540 (MH⁺, 100), 541(37), 542 (70), 543 (21), 544 (13).

[0238] The ketone 21b (35 mg, 0.11 mmol) and the primary amine 8 (40 mg,0.16 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (33 mg, 0.16 mmol) is added. The mixture is keptunder stirring at ambient temperature for several weeks. The reactionmedium is then washed with distilled water; the organic phase is driedand the solvent is evaporated to dryness (yield: 76%).

[0239] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.48 (d, 1H, H2′), 7.90 (d, 1H,H8′), 7.75 (d, 1H, H5′), 7.33 (dd, 1H, H6′), 6.33 (d, 1H, H3′), 6.02 (slarge, 1H, HN9′), 5.41 (d large, 1H, H6), 3.90 (d large, 1H, H5), 3.25(m, 2H, H₂C10′), 2.93 (m, 1H), 2.67 (m, 2H, H₂C11′), 2.45 (m, 3H), 2.20(m, 6H), 2.00 (m, 2H), 1.8−1.6 (m, 6H), 1.48 (m, 1H), 1.25 (m, 2H), 1.00(m, 9H).

[0240] MS (DCI/NH3+) m/z (%): 538 (10), 539 (9), 540 (MH⁺, 100), 541(40), 542 (40), 542 (67), 543 (23), 544 (14).

[0241] Production of Trioxaquine Dicitrates 25a and 25b

[0242] The trioxaquine 24a (63 mg, 0.12 mmol) is placed in solution inacetone (5 ml). Citric acid (70 mg, 3.0 equiv.) in solution in acetone(5 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0243] NMR ¹H (250 MHz, DMSO-d6) δ, ppm: 8.58 (d, 1H, H2′), 8.49 (d, 1H,H5′), 8.14 (s large, 1H, HN9′), 7.97 (d, 1H, H8′), 7.70 (dd, 1H, H6′),6.77 (d, 1H, H3′), 5.48 (d large, 1H, H6), 4.03 (d large, 1H, H5), 3.50(m, 2H, H₂C10′), 3.36 (m, 1H), 3.04 (m, 2H, H₂C13′), 2.75 (d, 4H,citrate), 2.65 (d, 4H, citrate), 2.60−1.95 (m, 9H), 1.90−1.45 (m, 10H),1.10 (m, 9H).

[0244] MS (ES) m/z (%):

[0245] in positive mode 540.3 (M⁺)

[0246] in negative mode 191.2 (citrate)

[0247] Elementary microanalysis: for C₄₃H₅₈O₁₇N₃Cl Theor. %: C 55.88 H6.33 N 4.55 Exper. %: C 55.35 H 6.27 N 4.37

[0248] The trioxaquine 24b (47 mg, 0.09 mmol) is placed in solution inacetone (4 ml). Citric acid (50 mg, 3.0 equiv.) in solution in acetone(4 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0249] NMR ¹H (250 MHz, DMSO-d6) δ, ppm: 8.58 (d, 1H, H2′), 8.45 (d, 1H,H5′), 8.01 (s large, 1H, HN9′), 7.95 (d, 1H, H8′), 7.69 (dd, 1H, H6′),6.75 (d, 1H, H3′), 5.47 (d large, 1H, H6), 4.05 (d large, 1H, H5), 3.48(m, 2H, H₂C10′), 3.35 (m, 1H), 3.03 (m, 2H, H₂C13′), 2.76 (d, 4H,citrate), 2.65 (d, 4H, citrate), 2.30 (m, 6H), 2.06 (m, 4H), 1.9−1.4 (m,9H), 1.09 (m, 9H).

[0250] MS (ES) m/z (%):

[0251] in positive mode 540.4 (M⁺)

[0252] in negative mode 191.0 (citrate)

[0253] Elementary microanalysis: for C₄₃H₅₈O₁₇N₃Cl, 1 H₂O Theor. %: C54.80 H 6.42 N 4.46 Exper. %: C 54.93 H 6.01 N 4.44

EXAMPLE 10

[0254] Trioxaquines 28a and 28b

[0255] Synthesis of Trioxanes Functionalised with a Ketone 26a and 26b

[0256] A mixture of 1,3-cyclohexadiene (400 mg, 5 mmol) andtetraphenylporphyrine (5 mg) in dichloromethane (10 ml) is irradiated inthe presence of molecular oxygen (1.15 bar) for 1 hour, at 5° C., with awhite bulb (200 W). The peroxide is obtained with a quantitative yield.The unprocessed peroxide in solution in dichloromethane is placed in abath at −70° C.; 4 molar equivalents of 1,4 cyclohexanedione (2.3 g, 20mmol) and 0.4 equivalent of trimethylsilyl trifluoromethane sulphonate(500 μl, 2.8 mmol) are added and the reaction mixture is kept understirring at −70° C. for 4 hours. The reaction is stopped by addingtriethylamine (1000 μl). After returning to ambient temperature, thereaction medium is washed with distilled water, dried on magnesiumsulphate and evaporated to dryness. Chromatography on silica column(hexane/ethyl acetate eluent, 70/30, v/v) is used to separate the twoisomer trioxanes 26a and 26b (overall yield: 2%).

[0257] Isomer 26a:

[0258] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 5.70 (m, 1H, H6), 5.57 (m, 1H,H7), 4.50 (m, 1H, H5), 4.25 (ddd, 1H, H10), 2.68 (m, 1H), 2.45 (m, 5H),2.32 (m, 2H), 2.03 (m, 2H), 1.90 (m, 1H), 1.55 m, 1H).

[0259] MS (DCI/NH3+) m/z (%): 241 (2), 242 (MNH₄ ⁺, 100), 243 (16), 244(5).

[0260] Isomer 26b:

[0261] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 6.00 (m, 1H), 5.73 (m, 1H), 4.50(m, 1H), 4.20 (m, 1H), 2.60−1.80 (m, 12H).

[0262] MS (DCI/NH3+) m/z (%): 241 (2), 242 (MNH₄ ⁺, 100), 243 (17), 244(5).

[0263] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquines 27a and 27b

[0264] The ketone 26a (9 mg, 0.04 mmol) and the primary amine 1 (12 mg,0.05 mmol) are placed in solution in CH₂Cl₂ (3 ml). Sodiumtriacetoxyborohydride (21 mg, 0.10 mmol) is added. The mixture is keptunder stirring at ambient temperature for 15 hours. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 35%).

[0265] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.49 (d, 1H, H2′), 7.93 (2×d, 1H,H8′), 7.75 (2×d, 1H, H5′), 7.37 (m, 1H, H6′), 6.35 (m, 2H, H3′ andHN9′), 5.66 (d large, 1H, H6), 5.55 (d large, 1H, H7), 4.48 (d large,1H, H5), 4.15 (d large, 1H, H10), 3.38 (m, 2H, H₂C10′), 3.10 (m, 2H,H₂C11′), 2.67 (m, 2H), 2.49 (m, 3H), 2.30 (m, 2H), 2.10−1.30 (m, 7H).

[0266] MS (DCI/NH3+) m/z (%): 430 (MH⁺, 100), 431 (30), 432 (47).

[0267] The ketone 26b (7 mg, 0.03 mmol) and the primary amine 1 (15 mg,0.07 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (12 mg, 0.06 mmol) is added. The mixture is keptunder stirring at ambient temperature for one week. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 45%).

[0268] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (2×d, 1H, H2′), 7.92 (2×d,1H, H8′), 7.70 (2×d, 1H, H5′), 7.36 (2×dd, 1H, H6′), 6.34 (2×d, 1H,H3′), 6.06 (s large, 1H, HN9′), 5.99 (m, 1H, H6), 5.72 (m, 1H, H7), 4.41(m, 1H), 4.10 (m, 1H), 3.32 (m, 2H, H₂C10′), 3.05 (m, 2H, H₂C11′), 2.63(m, 2H), 2.30 (m, 4H), 2.10 (m, 1H), 2.00−1.35 (m, 7H).

[0269] MS (DCI/NH3+) m/z (%): 428 (15), 429 (9), 430 (MH⁺, 100), 431(30), 432 (49), 433 (14).

[0270] Production of Trioxaquine Dicitrate 28b

[0271] The trioxaquine 27b (6 mg, 0.014 mmol) is placed in solution inacetone (1 ml). Citric acid (10 mg, 3.7 equiv.) in solution in acetone(1 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0272] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.65 (d, 1H, H2′), 8.37 (d, 1H,H5′), 7.98 (d, 1H, H8′), 7.69 (dd 1H, H6′), 6.75 (d, 1H, H3′), 6.05 (m,1H), 5.80 (m, 1H), 4.57 (m, 1H), 4.30 (m, 1H), 3.75 (m, 2H, H₂C10′),3.34 (m, 3H, H₂C13′ and 1H), 2.80 (d, 4H, citrate), 2.68 (d, 4H,citrate), 2.50−1.50 (m, 12H).

[0273] MS (ES) m/z (%):

[0274] in positive mode 430.2 (M⁺)

[0275] in negative mode 191.2 (citrate)

[0276] Elementary microanalysis: for C₃₅H₄₄O₁₇N₃Cl Theor. %: C 51.65 H5.45 N 5.17 Exper. %: C 51.69 H 5.18 N 4.66

EXAMPLE 11

[0277] Trioxaquine 32

[0278] Synthesis of Oxoaldehyde 4-oxopentanal 29

[0279] To a suspension of pyridinium chlorochromate PCC (6.4 g, 30 mmol)in dichloromethane (25 ml) 3-acetylpropan-1-ol (2.0 g, 20 mmol) isslowly added. The reaction mixture is left under stirring at ambienttemperature for 2 hours and is then filtered on a silica gel with ether.The black residue is washed twice with ether and the filteredether-treated phases are pooled and evaporated. The aldehyde is obtainedin the form of dark liquid (80% purity, 84% yield).

[0280] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 9.75 (S, 1H), 2.70 (s large, 4H),2.14 (s, 3H).

[0281] Synthesis of Trioxane Functionalised with a Ketone 30

[0282] A mixture of 2,3-dimethylbut-2-ene (270 mg, 3.2 mmol) andtetraphenylporphyrine (5 mg) in dichloromethane (5 ml) is irradiated inthe presence of molecular oxygen (1.15 bar) for 7 hours, at 5° C., witha white bulb (200 W). The peroxide is obtained with a quantitativeyield. The unprocessed peroxide in solution in dichloromethane is placedin a bath at −70° C.; 10 molar equivalents of 4-oxopentanal 29 (2.2 mg,22 mmol) and a few drops of trifluoroacetic acid CF₃COOH are added. Themixture is stirred at ambient temperature for 90 minutes and then 2equivalents of N-iodosuccinimide (1.35 g, 6 mmol) are added and themixture is kept under stirring and protected from light for 3 hours,after which the reaction medium is washed with 20% sodium thiosulphatefollowed by distilled water. After drying on sodium sulphate andevaporation to dryness, the functionalised trioxane 30 is purified bychromatography on alumina column (hexane/ether eluent, 50/50, v/v)(yield: 14%).

[0283] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 5.39 (t, 1H, H3), 3.31 (d,²J_(HH)=11 Hz, 1H, HC10), 3.12 (d, ²J_(HH)=11 Hz, 1H, HC10), 2.55 (m,2H, H₂C12), 2.15 (s, 3H, H₃C14), 1.83 (m, 2H, H₂C11), 1.48 (2 s, 6H,H₃C8 and H₃C7), 1.06 (s, 3H, H₃C9).

[0284] MS (DCI/NH3+) m/z (%): 359 (22), 360 (MNH₄ ⁺, 100), 361 (14), 362(2).

[0285] Coupling of Primary Amine with the ketone by Reductive Amination:Production of Trioxaquine 31

[0286] The ketone 30 (13 mg, 0.04 mmol) and the primary amine 1 (15 mg,0.07 mmol) are placed in solution in CH₂Cl₂ (4 ml). Sodiumtriacetoxyborohydride (11 mg, 0.05 mmol) is added. The mixture is keptunder stirring at ambient temperature for 7 days. The reaction medium isthen washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 59%).

[0287] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (d, 1H, H2′), 7.95 (d, 1H,H8′), 7.72 (d, 1H, H5′), 7.39 (2×dd, 1H, H6′), 6.37 (2×d, 1H, H3′), 6.00(s large, 1H, HN9′), 5.37 (t, 1H, H3), 3.30 (m, 3H, H₂C10′ and HC10),3.11 (d, ²J_(HH)=11 Hz, 1H, HC10), 3.00 (m, 2H, H₂C11′), 2.74 (m, 1H,HC13), 1.8 (s large, 1H, HN12′), 1.65−1.55 (m, 4H, H₂C11 and H₂C12),1.50 (2 s, 6H, H₃C8 and H₃C7), 1.10 (d, 3H, H₃C14), 1.05 (s, 3H, H₃C9).

[0288] MS (DCI/NH3+) m/z (%) : 547 (6), 548 (MH⁺, 100), 549 (29), 550(37), 551 (9).

[0289] Production of Trioxaquine Dicitrate 32

[0290] The trioxaquine 31 (40 mg, 0.073 mmol) is placed in solution inacetone (1 ml). Citric acid (52 mg, 3.7 equiv.) in solution in acetone(1 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0291] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.63 (d, 1H, H2′), 8.37 (d, 1H,H5′), 7.99 (d, 1H, H8′), 7.71 (dd, 1H, H6′), 6.77 (d, 1H, H3′), 5.37 (t,1H, H3), 3.7 (m, 4H, H₂C10′, HC10 and HC13), 3.34 (m, 3H, H₂C11′ andHC10), 2.78 (d, 4H, citrate), 2.67 (d, 4H, citrate), 2.0−1.6 (m, 4H),1.54−1.35 (m, 6H), 1.22 (m, 6H).

[0292] MS (ES) m/z (%):

[0293] in positive mode 548.2 (M⁺)

[0294] in negative mode 191.2 (citrate)

[0295] Elementary microanalysis: for C₃₄H₄₁O₁₇N₃Cl Theor. %: C 43.81 H5.08 N 4.51 Exper. %: C 44.08 H 4.69 N 4.72

EXAMPLE 12

[0296] Trioxaquine 34

[0297] Coupling of Primary Amine with the Ketone by Reductive Amination:Production of Trioxaquine 33

[0298] The ketone 30 (23 mg, 0.07 mmol) and the primary amine 8 (27 mg,0.11 mmol) are placed in solution in CH₂Cl₂ (5 ml). Sodiumtriacetoxyborohydride (27 mg, 0.13 mmol) is added. The mixture is keptunder stirring at ambient temperature for 10 days. The reaction mediumis then washed with distilled water; the organic phase is dried and thesolvent is evaporated to dryness (yield: 10%).

[0299] NMR ¹H (250 MHz, CDCl₃) δ, ppm: 8.50 (d, 1H, H2′), 7.90 (d, 1H,H8′), 7.75 (d, 1H, H5′), 7.34 (2×dd, 1H, H6′), 6.38 (2×d, 1H, H3′),6.00−5.70 (s large, 1H, HN9′), 5.35 (t, 1H, H3), 3.30 (m, 3H, H₂C10′ andHC10), 3.11 (d, ²J_(HH)=11 Hz, 1H, HC10), 2.71 (m, 2H, H₂C13′), 2.51 (m,1H, HC13), 1.78 (m, 4H), 1.65 (m, 4H), 1.48 (2 s, 6H, H₃C8 and H₃C7),1.25 (m, 3H), 1.09 (s, 3H, H₃C9). M

[0300] S (DCI/NH3+) m/z (%): 576 (MH⁺).

[0301] Production of Trioxaquine Dicitrate 34

[0302] The trioxaquine 33 (20 mg, 0.035 mmol) is placed in solution inacetone (1 ml). Citric acid (24 mg, 4.5 equiv.) in solution in acetone(1 ml) is added. The trioxaquine dicitrate precipitates; it iscentrifuged, washed twice in diethyl ether and vacuum-dried.

[0303] NMR ¹H (250 MHz, DMSO-d₆) δ, ppm: 8.62 (d, 1H, H2′), 8.46 (d, 1H,H5′), 7.96 (m, 2H, H8′ and HN9′), 7.70 (m, 1H, H6′), 6.73 (d, 1H, H3′),5.50 (t, 1H, H3), 4.0−3.0 (m, 7H, H₂C10′, H₂C13, H₂C10′ and HC13), 2.77(d, 4H, citrate), 2.66 (d, 4H, citrate), 1.9−1.6 (m, 8H), 1.53 (m, 6H),1.4−1.1 (m, 6H).

[0304] MS (ES) m/z (%) : in positive mode 576.2 (M⁺)

EXAMPLE 13

[0305] Study of the Anti-Malarial Activity of Trioxaquines on P.falciparum.

[0306] The in vitro results obtained on P. falciparum cultured in humanred blood cells are given below.

[0307] Experimental Section

[0308] The P. falciparum strains are cultured continuously according toTrager and Jensen's method 5, with the following modifications 6: theparasites are maintained in human red blood cells (O±), diluted to 1%haematocrit in RPMI 1640 medium supplemented with 25 mM Hepes+30 mMNaHCO₃ and complemented with 5% AB+human serum. The parasite populationsare synchronised over a 4 hour period by flotation with a gelatinesolution, followed by lysis with 5% D-sorbitol 7,8. The Nigerian strainis considered to be susceptible to chloroquines and the FcM29-Cameroonand FcB1-Columbia strains are chloroquine-resistant (IC₅₀ forchloroquine >100 nM) 9,10. The anti-malarial activity tests areperformed using Desjardins' radioactive micromethod 11. The tests areperformed in triplicate, in 96-well microplates, the readings being at1% haematocrit and 0.5-1% parasitaemia. For each test, the parasites areincubated with decreasing concentrations of drug for either 32 hours and72 hours (4 wells contain chloroquine diphosphate as a reference). Thefirst dilution of the drug is performed at 10 mg/ml indimethylsulphoxide and the following dilutions are made with RPMI 1640.The parasite growth is measured with the incorporation of tritiatedhypoxanthine compared to incorporation in the absence of drug (taken tobe 100%). 12 and the IC₅₀ values are determined graphically by plottingthe percentage of inhibition as a function of the drug concentration.The IC₅₀ values measured after 32 hours, equivalent to the end of thetrophozoite stage, are used to evaluated the influence of the compoundon parasite maturation, the IC₅₀ values measured after 72 hours,corresponding to 1.5 of the life cycle in red blood cells, indicate apossible effect on erythrocyte reinvasion.

[0309] Trioxaquine Structures Tested:

[0310] Results

[0311] Trioxaquines 9, 3 and 6 and trioxaquine citrate 4 were testedindependently on the Nigerian, FcB1 and FcM29 strains; the resultsobtained were compared to those of chloroquine diphosphate and to thoseof two trioxaquine fragments: quinoline-amine 1 (n=2) andtrioxane-ketone 2.

[0312] The results are summarised in the following table:

[0313] Table. IC50 values measured for 9, 3, 6, 4 tested independentlyon three Plasmodium falciparum strains. FcB1- FcM29- Colombia CameroonNigerian (CQR)^(a) (CQR+)^(a) (CQS)^(a) 32 72 32 72 32 72 hrs hrs hrshrs hrs hrs 6 ng/ml 40 35 25 10 50 50 (n = 3) nM 69 60 43 17 86 86 9ng/ml 16 10 22 20 20 20 (n = 4) nM 27 17 37 34 34 34 3 ng/ml 15 5 18 101 1 (n = 2) nM 26 9 32 18 1.8 1.8 4 ng/ml 35 20 n.d.^(b) n.d.^(b) 33 7(n = 2) , citrate nM 37 21 35 8 Quinoline- ng/ml 35 20 n.d.^(b) n.d.^(b)33 7 amine1 Trioxane- nM 113 36 n.d.^(b) n.d.^(b) 149 45 ketone 2Chloroquine ng/ml 75 60 100 80 45 10 diphosphate^(c) nM 145 116 194 15587 19

[0314] highly chloroquine resistant strain, CQS=chloroquine susceptiblestrain. ^(b) Not determined. ^(c) Given for comparison purposes.

[0315] The IC₅₀ values obtained for the compounds 3, 4, 6 and 9 arebetween 5 and 50 ng/ml, which is equivalent to a concentration of 8 to86 nM.

[0316] All the trioxaquine samples are more active than chloroquine, onboth susceptible and resistant strains (except for 6 on the Nigeriansusceptible strain). The high efficacy of 9, 3 and 4 on the susceptibleand resistant strains, significantly greater than the activity of notonly chloroquine, but also of each of the two fragments, quinoline-amine1 (n=2) and trioxane-ketone 2, indicates a significant synergic effectbetween the trioxane and chloroquinoline fragments of these compounds.

[0317] The citrate form of compound 4 increases its solubilityconsiderably in aqueous media while retaining a high activity.Protonation by other acids than citric acid produces salts offering thesame advantages (hydrochloride, sulphate, tartrate).

[0318] The low IC₅₀ values obtained with trioxaquine 3 on the FcB1,FcM29 strains and on the Nigerian strain (9 nM, 18 nM and 1.8 nMrespectively) and with 9 and the citrate 4, indicate that the efficacyof these compounds is retained for a wide spectrum of parasites.

EXAMPLE 14

[0319] Preparation of Pharmaceutical Formulations Based on MoleculesAccording to the Invention.

[0320] in the form of scored tablets

[0321] active ingredient: 100 mg

[0322] excipients: starch, hydrated silica, amylaceous ground sugar,gelatine, magnesium stearate.

[0323] in the form of film-coated tablets

[0324] active ingredient: 300 mg

[0325] excipients: Core: wheat starch, amylaceous ground sugar (sucrosepowder supplemented with starch), hydrated silica, gelatine, magnesiumstearate. Coating: methylhydroxy propylcellulose, poloxyethyleneglycol20,000.

[0326] in the form of syrups

[0327] active ingredient: 25 mg/ml

[0328] excipients: citric acid, sucrose, coffee extract, caramel,purified water.

[0329] in the form of solutions for injection

[0330] active ingredient: 100 mg for one 1 ml ampoule, 200 mg for one 2ml ampoule, 400 mg for one 4 ml ampoule.

[0331] excipients: sodium chloride and water for injections.

[0332] in the form of 25% solutions for injection

[0333] active ingredient: 500 mg

[0334] excipients: lactic acid (solubilising agent), formic acid, waterfor injections.

[0335] Preservative: anhydrous sodium sulphite equivalent to 0.61 mgsulphur anhydride/amp.

[0336] in the form of 100 mg/ml oral suspensions:

[0337] active ingredient: 20 mg/ml.

[0338] excipients: microcrystalline cellulose and sodiumcarboxymethylcellulose, propylene glycol, sorbitol, anhydrous citricacid, sodium citrate, sodium benzoate, banana-vanilla flavour,dimethylpolysiloxane emulsion, purified water.

EXAMPLE 15

[0339] Activity of trioxaquine-citrate 4 on human isolates

[0340] Trioxaquine-citrate 4, referred to as DU-1102, was tested onhuman Plasmodium falciparum isolates, some of which were chloroquine-and/or pyrimethamine-resistant. The median inhibitory concentrationvalues, IC50, obtained are 11 to 68 ng/ml, corresponding to a geometricmean of 41 ng/ml, i.e. 43 nM.

[0341] The activity of DU-1102 on these isolates is independent of theirsusceptibility or their resistance to the other anti-malarial agentstested.

[0342] There is no correlation between DU-1102 on the one hand andchloroquine or pyrimethamine on the other, which indicates the absenceof the probability of cross-resistance between DU-1102 and saidanti-malarial agents already used.

[0343] These results indicate the satisfactory efficacy of trioxaquineson wild P. falciparum strains.

[0344] Activity of trioxaquine-citrate DU-1302, represented below:

[0345] 2.1. In vitro, this compound shows an activity, LC50=6 nM on P.falciparum in culture.

[0346] 2.2. The trioxaquine citrate DU-1302 is active in vivo in miceinfected with P. vinckei (“14 day” test: treatment for 4 consecutivedays starting 24 hours after inoculation of the parasite).

[0347] ED50=5 mg/kg/day, or 6 μmol/kg/day, by the intraperitoneal route,with no observable re-emergence for 2 months,

[0348] ED50=18 mg/kg/day, or 20 μmol/kg/day, by the oral route.

[0349] 2.3. The trioxaquine-citrate DU-1302 does not show any apparenttoxicity after administration to healthy mice, at a dose of 100mg/kg/day by the oral route, for three consecutive days.

[0350] 3. Absence of mutagenicity of trioxaquine-citrates DU-1102 andDU-1302

[0351] The above two compounds do not induce DNA repair (no SOS responsein E. coli GE864 at concentrations of 5, 10, 15 and 20 μM: the controlused is 3 μM mitomycin C). Therefore, they are non-mutagenic for E. coliat these concentrations.

[0352] Formulas of compounds for which the synthesis is described in theexamples

1. Dual molecules according to the invention characterised in that theyconsist of coupling products complying with the formula (I)

wherein A represents a residue of molecule with anti-malarial activity,Y₁ and Y₂, identical or different, represent a linear or ramifiedalkylene chain at C1 to C5, containing if applicable one or more amine,amide, sulphonamide, carboxyl, hydroxyl, ether or thioether radicals,said alkylene chain at C1 to C5 being substituted if applicable by analkyl radical at C1 to C5, with the possibility of either Y₁ or Y₂ beingabsent, U is an amine, amide, sulphonamide, carboxyl, ether or thioetherfunction, said function linking Y, and Y₂, Z₁ and Z₂, identical ordifferent, represent a saturate or unsaturated, linear, ramified orcyclic arylene or alkylene radical, with the possibility of either Z₁ orZ₂ being absent, or Z₁+Z₂ together represent a polycyclic structureincluding the junction carbons Ci and Cj, R₁ and R₂, identical ordifferent, represent a hydrogen atom or a functional group capable ofincreasing the hydrosolubility of the dual molecule, advantageouslyselected from —COOH, —OH, —N(R_(a), R_(b)) where R_(a) and R_(b),identical or different, represent a hydrogen atom or an alkyl radical atC1 to C5, R_(x) and R_(y) form a cyclic peroxide with 4 to 8 chainlinks, which may comprise 1 or 2 additional oxygen atoms in the cyclicstructure, Cj being one of the peaks of said cyclic peroxide, or R_(x)or R_(y) is a cyclic peroxide with 4 to 8 chain links, which maycomprise one or more1 or 2 additional oxygen atoms in the cyclicstructure, and one or more substituents R₃, identical or different,occupying any separate positions on the cycle, at least one representinga halogen atom, an —OH group, a —CF₃ group, an aryl radical, an alkyl oralkoxy radical at C1 to C5, an —NO₂ group, the other substituent(s)having one of these correspondences or a hydrogen atom, with thepossibility of substituting the carbonated peaks of the cyclic peroxideif applicable by one or more substituents as defined for R₃, with thepossibility of two adjacent substituents forming a cyclic structure with5 to 6 chain links, saturated or unsaturated, if applicable substitutedby one or more substituents R₃ in any position, with the possibility ofthe other substituent R_(x) or R_(y) being R₃, and their addition saltswith pharmacological acceptable acids.
 2. Molecules according to claim1, characterised in that A represents a nitrous heterocycle selectedfrom an aminoquinoline according to formula (II) or a 1,5-naphtyridineaccording to formula (III) below

wherein R₃ represents one or more identical or different substituentsoccupying separate positions, at least one representing a halogen atom,an —OH group, a —CF₃ group, an aryl radical, an alkyl or alkoxy radicalat C1 to C5, an —NO₂ group, the other substituent(s) having one of thesecorrespondences or a hydrogen atom, R₄ represents a linear, ramified orcyclic alkyl radical at C1 to C5, or a hydrogen atom.
 3. Moleculesaccording to claim 1, characterised in that, A represents a radicalaccording to formula (IV) R₅—CHOH—  (IV) wherein R₅ represents an arylradical or a nitrous heterocyclic residue.
 4. Molecules according toclaim 3, characterised in that R₅ is a 9-phenanthrenyl or 4-quinoleinylradical, possibly substituted by one or more R₃ groups.
 5. Moleculesaccording to claim 1, characterised in that A represents aphenol-2(aminomethyl) residue according to formula (V)

wherein R₃, R_(a) and R_(b) are as defined in claim
 1. 6. Moleculesaccording to claim 1, characterised in that A represents a biguanideresidue selected from proguanil derivatives according to formula (VI)

or cycloguanil according to formula (VII)

wherein R₃ is as defined in claim
 1. 7. Molecules according to claim 1,characterised in that A represents a residue of pyrimidine and moreparticularly of pyrimethamine according to formula (VIII)

or formula (IX)

wherein R₃ is as defined in claim
 1. 8. Molecules according to claim 1,characterised in that A represents an acridine residue according toformula (X)

wherein R₃ and R₄ are as defined in claims 1 and 2, respectively. 9.Molecules according to any of claims 1 to 8, characterised in that R_(x)and R_(y) form a cyclic peroxide together.
 10. Molecules according toclaim 9, characterised in that R_(x) and R_(y) represent a trioxanesubstituted by one or more substituents R₃.
 11. Molecules according toany of claims 1 to 10, characterised in that R₃ represents a singlesubstituent, said substituent being a halogen atom selected from F, Cl,Br, I or 2 substituents occupying separate positions, one representing ahalogen atom selected from F, Cl, Br, I, and the other an alcoxy group.12. Molecules according to any of claims 1 to 11, characterised in thatZ₁ and Z₂ represent a cyclohexyl or bi-cyclopentyl radical.
 13. Methodto prepare dual molecules according to claim 1 wherein A is anamino-quinoline and R_(x) and R_(y) form a trioxane, characterised inthat a compound according to formula (XI)

wherein R₃ is as defined above and “hal” represents a halogen atom, isreacted with a diamine derivative according to formula (XII) R₄—NH—Y₁—U₁  (XII) where R₄ and Y₁ are as defined above and U₁ represents an —NH₂group, producing a compound according to formula (XIII)

wherein R₃, R₄ and Y₁ are as defined above, b) -irradiation in thepresence of molecular oxygen and a photosensitising agent, of aderivative according to formulas (XIV) to (XVII) below

followed by the reaction with a diketone, such as 1,4-cyclohexadioneaccording to formula (XVIII) or cis-bicyclo(3.3.0]octane-3,7-dioneaccording to formula (XIX)

producing trioxanes functionalised with a ketone, according to thegeneral formula (XX)

is wherein Z₁, Z₂ and R₃ are as defined above, c) -coupling of thederivative according to formula (XIII) with the trioxane according toformula (XX), by reductive amination, followed if applicable by areaction with a pharmaceutically acceptable acid, to obtain the couplingproduct in salt form.
 14. Pharmaceutical formulations characterised inthat they comprise an effective quantity of at least one couplingproduct as defined in any of claims 1 to 12, associated with apharmaceutically inert vehicle.
 15. Pharmaceutical formulationsaccording to claim 14, which may be administered by the oral, rectal orinjectable route.
 16. Formulations according to claim 15, characterisedin that they comprise 10 to 100 mg of active ingredient per dosage unitfor oral administration.
 17. Formulations according to claim 16,characterised in that, for oral administration, they are presented inthe form of tablets, pills, capsules or drops.
 18. Formulationsaccording to claim 15, characterised in that they comprise 10 to 50 mgof active ingredient per dosage unit for administration by injection.19. Formulations according to claim 15, characterised in that, foradministration by injection, they are presented in the form of solutionsfor injection by the intravenous, subcutaneous or intramuscular route,produced from sterile or sterilisable solutions, or suspensions oremulsions.
 20. Pharmaceutical formulations according to claim 14 or 15intended for the treatment of malaria.
 21. Use of dual moleculesaccording to any of claims 1 to 12 to produce medicinal products with ananti-malarial activity.